Monoclonal antibody binding affinity determined by microchip‐based capillary electrophoresis

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Abstract
The affinity constant of a monoclonal antibody to fluorescently labeled bovine serum albumin (BSA*) was measured in diluted mouse ascites fluid using a microfluidic chip to perform affinity capillary electrophoresis. Borofloat glass-based devices could be used repeatedly with samples for many months. On-chip separations were performed in less than 60 s, and 30–60 s was required for manual sample exchange. The change in peak height for BSA* with increasing BSA*/anti-BSA concentration ratio was used to determine concentration changes in bound and free BSA*. A Scatchard plot analysis gave an affinity constant (more exactly the intrinsic association constant) of 3.5 ± 0.6 × 107 M−1 for a 1:1 stoichiometric ratio. Two affinity complexes were separated. One complex was identified by the Scatchard method as having a 1:1 stoichiometric ratio. The other complex is proposed to have a stoichiometry with an excess of anti-BSA to BSA*, most likely (anti-BSA)2-BSA*, on the basis of a faster migration time than the 1:1 complex, a decrease in the amount of this complex with increasing [BSA*], and predictions of theoretical models for multi-valent antigens. Potential applications of microchip-based devices in affinity measurements are discussed.